TY - JOUR
T1 - Independently melting modules and highly structured intermodular junctions within complement receptor type 1
AU - Kirkitadze, Marina D.
AU - Krych, Malgorzata
AU - Uhrin, Dusan
AU - Dryden, David T.F.
AU - Smith, Brian O.
AU - Cooper, Alan
AU - Wang, Xuefeng
AU - Hauhart, Richard
AU - Atkinson, John P.
AU - Barlow, Paul N.
PY - 1999/6/1
Y1 - 1999/6/1
N2 - A segment of complement receptor type 1 (CR1) corresponding to modules 15-17 was overexpressed as a functionally active recombinant protein with N- glycosylation sites ablated by mutagenesis (referred to as CR1~15-17-). A protein consisting of modules 15 and 16 and another corresponding to module 16 were also overexpressed. Comparison of heteronuclear nuclear magnetic resonance (NMR) spectra for the single, double, and triple module fragments indicated that module 16 makes more extensive contacts with module 15 than with module 17. A combination of NMR, differential scanning calorimetry, circular dichroism, and tryptophan-derived fluorescence indicated a complex unfolding pathway for CR1~15-17-. As temperature or denaturant concentration was increased, the 16-17 junction appeared to melt first, followed by the 15-16 junction, and module 17 itself; finally, modules 15 and 16 became denatured. Modules 15 and 16 adopted an intermediate state prior to total denaturation. These results are compared with a previously published study [Clark, N. S., Dodd, I, Mossakowska, D. E., Smith, R. A. G., and Gore, M. G. (1996) Protein Eng. 9, 877-884] on a fragment consisting of the N- terminal three CR1 modules which appeared to melt as a single unit.
AB - A segment of complement receptor type 1 (CR1) corresponding to modules 15-17 was overexpressed as a functionally active recombinant protein with N- glycosylation sites ablated by mutagenesis (referred to as CR1~15-17-). A protein consisting of modules 15 and 16 and another corresponding to module 16 were also overexpressed. Comparison of heteronuclear nuclear magnetic resonance (NMR) spectra for the single, double, and triple module fragments indicated that module 16 makes more extensive contacts with module 15 than with module 17. A combination of NMR, differential scanning calorimetry, circular dichroism, and tryptophan-derived fluorescence indicated a complex unfolding pathway for CR1~15-17-. As temperature or denaturant concentration was increased, the 16-17 junction appeared to melt first, followed by the 15-16 junction, and module 17 itself; finally, modules 15 and 16 became denatured. Modules 15 and 16 adopted an intermediate state prior to total denaturation. These results are compared with a previously published study [Clark, N. S., Dodd, I, Mossakowska, D. E., Smith, R. A. G., and Gore, M. G. (1996) Protein Eng. 9, 877-884] on a fragment consisting of the N- terminal three CR1 modules which appeared to melt as a single unit.
UR - http://www.scopus.com/inward/record.url?scp=0033153280&partnerID=8YFLogxK
U2 - 10.1021/bi982453a
DO - 10.1021/bi982453a
M3 - Article
C2 - 10353813
AN - SCOPUS:0033153280
SN - 0006-2960
VL - 38
SP - 7019
EP - 7031
JO - Biochemistry
JF - Biochemistry
IS - 22
ER -